BHB-Citrate Combination Products for Renal Health and Treating Disease

ABSTRACT

Compositions and methods for supporting health, especially renal health, comprising ketonic agents that recapitulate beneficial effects of ketosis by exogenously administered agents. The agents include BHB, analogs thereof, and GPR109A agonists. The agents may further include crystal precipitation inhibitors which synergistically improve treatment of certain renal conditions. The agents may be used in dietary supplements and therapeutic compositions for the treatment of cystic kidney diseases such as polycystic kidney disease, ciliopathies, and other conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/819,079, entitled “Methods and Compositions for Supporting RenalHealth,” filed Mar. 14, 2020, which claims the benefit of priority toU.S. Provisional Patent Application Ser. No. 62/818,538 entitled “BHBand BHB-Citrate Combination Therapy for Polycystic Kidney Disease,”filed Mar. 14, 2019 the contents of which applications are herebyincorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberR01-DK109563 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Renal disease is a major health issue. Among the various renalconditions that afflict people, renal cysts are present in numerouspathologies of the kidney. Polycystic kidney disease (PKD) is anespecially serious condition with high morbidity and mortality.Autosomal dominant polycystic kidney disease (ADPKD) is considered themost common life-threatening monogenic disease. The PKD disease state ischaracterized by the growth of fluid-filled cysts in the kidneys whichprogressively enlarge, leading to destruction of normal renal tissue andfunction. Renal cyst growth in PKD is driven by several growth factors,hormones and cytokines that are present in the cyst fluid and whichstimulate the cyst-lining epithelial cells. In the United States, theonly FDA-approved therapeutic for ADPKD is the vasopressin receptor 2antagonist tolvaptan. Its use is complicated due to significant sideeffects and potential toxicities, poor cost-effectiveness and questionspotentially limited efficacy. Accordingly, there remains a need in theart for effective ways of preventing and treating renal conditions,including cystic renal conditions such as polycystic kidney disease.

Polycystic kidney disease is one of several pathological conditionsclassified as a ciliopathy. Cilia are microtubule-containing structuresthat protrude from the surface of most cells types. Cilia, especiallyimmotile primary cilia, act as a “cellular antenna” with variousfunctions including sensory and cell-to-cell communication roles. Manypathological conditions are associated with mutations that result inabnormal cilial formation or function. There are over 35 knownciliopathies, and for many of these conditions, effective treatments arenot available. Accordingly, there remains a need in the art for novelways of preventing and ameliorating ciliopathies.

Meanwhile, the metabolic state of ketosis is a growing area of study.Ketosis is characterized by elevated abundance of ketone bodies in bloodand other compartments. Ketosis is a metabolic pathway induced by lowglucose conditions, for example, being induced by low-carbohydrate dietor fasting. Numerous health benefits appear to be associated withdietary ketosis, including weight loss and appetite control, improvedcholesterol status, and stabilization of blood glucose and insulin. Theuse of ketogenic diets has been applied to control epilepsy in childrenand blood glucose in Type 2 diabetics. It is thought that ketogenicdiets may be of benefit in addressing many other conditions, forexample, in the context of kidney injury in surgical subjects, caloricrestriction was found to have protective effect on renal function, asdescribed in Grundmann et al., Preoperative Short-Term CalorieRestriction for Prevention of Acute Kidney Injury After Cardiac Surgery:A Randomized, Controlled, Open-Label, Pilot Trial. J Am Heart Assoc 7,(2018.) However, the mechanisms by which ketosis acts on pathologicalprocesses and conditions is not well understood, and previouslyrecognized dietary benefits have not been translated to practicalapplications and interventions. Accordingly, there is a need in the artfor an improved understanding of the molecular signaling mechanisms orother physiological effects by which ketosis achieves health benefits.Furthermore, long-term adherence to ketogenic diets is difficult andcompliance is poor for many, if not most, subjects. Accordingly, thereis further need in the art for novel therapies that harness thebeneficial effects of ketosis while being readily administered orapplied.

SUMMARY OF THE INVENTION

By various novel discoveries, the inventors of the present disclosurehave provided the art with novel compositions of matter and methods forimproving health and preventing and treating disease. The inventors ofthe present disclosure have elucidated that beta hydroxybutyrate (BHB),produced in dietary ketosis, activates certain beneficial pathways andprocesses. Among these, the activation of GPR109A by BHB is shown hereinto provide various therapeutic effects. Based on these discoveries, theinventors of the present disclosure have identified and developedcompositions with beneficial and therapeutic properties, and methods ofapplying them.

In a first aspect, the scope of the invention encompasses novelcompositions for promoting health, preventing disease, and treatingpathological conditions. These compositions, termed “ketoniccompositions” herein, comprise BHB, BHB precursors that convert to BHBin vivo, BHB-like molecules, and functional mimics of BHB, includingchemically unrelated species, that recapitulate the beneficial signalingactivities of BHB.

Advantageously, the ketonic compositions can be formulated as dietarysupplements, for convenient oral administration. In many cases, theketonic compositions comprise inexpensive off-the-shelf ingredients withlong-established safety profiles, and thus can be provided without theneed for clinical trials, and may be utilized without the need for aprescription.

In another aspect, by the discoveries herein, the scope of the inventionencompasses novel uses of GPR109A agonists. Many such agents have beenpreviously developed and clinically investigated for other purposes, andmay be repurposed in the methods of the invention for novel therapeuticapplications.

In another aspect, the scope of the invention encompasses novelcompositions and methods for improving renal health, and preventing andtreating various conditions of the kidney. In one aspect, the inventionsdisclosed herein are advantageously applied for subjects at risk of orsuffering from various renal cystic conditions, especially polycystickidney disease. The scope of the invention encompasses dietarysupplements and uses thereof for promoting renal health by preventingthe formation of cysts and selectively destroying cyst epithelial cells.

Likewise, in another novel use, ketonic agents may be used to prevent orameliorate the severity of acute kidney injury, for example, being usedas preventative agents prior to surgery to reduce the risk or severityof acute kidney injury associated with surgery. In another novel use,ketonic agents may be used to ameliorate the risk or severity ofischemic perfusion injury, for example, as preventative, e.g.,presurgical treatments.

In another aspect, the inventors of the present disclosure havedetermined that the ketonic agents of the invention may be combined withcrystal precipitation inhibitors with synergistic beneficial activity.By preventing the formation of calcium, oxalate, or uric acid crystals,which underlie or exacerbate the pathology of various renal conditionssuch as cysts, acute kidney injury, and reperfusion injury, thecombination products of the invention provide enhanced preventative andtherapeutic effects. Specifically, citrate, a safe and readily availableingredient, is a crystal precipitation inhibitor that can be combinedwith ketonic agents, for example, in dietary supplements.

In another aspect, the inventors of the present disclosure havedeveloped novel compositions of matter comprising bi-functionalmolecules made up of a ketonic agent and a crystal precipitationinhibitor, for example, a BHB-citrate combined molecule. These novelcombination products provide the art with synergistic beneficial effectsfrom a single agent having favorable formulation and administrationproperties.

In another aspect, the inventors of the present disclosure havedetermined that cystic epithelial cells are metabolically inflexible,being dependent upon glucose and being unable to metabolize fatty acids.Accordingly, in one aspect, the scope of the invention encompasses novelcombination products made up of ketonic agents in combination withcompositions that are lipotoxic to pathological cells such as polycystickidney cyst epithelial cells and other undesirable cell types. Theco-administration of ketonic agents and lipotoxic compositions providesenhanced preventatives and treatments for PKD cysts and otherconditions.

In another aspect, the scope of the invention encompasses novel means ofpreventing and treating ciliopathies. The ketonic compositions of theinvention, including exogenous ketones and GPR109A agonists, may beutilized in dietary supplements and therapeutic compositions forameliorating the risk, onset, and severity of numerous ciliopathies suchas PKD and retinitis pigmentosa.

These various inventions, and other beneficial applications of thediscoveries disclosed herein, address a number of unmet needs in theart, as described in more detail in the sections that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of the effects of a ketogenicdiet or the application of ketonic compositions on polycystic kidneys.Without intervention, kidneys are ridden with cysts and cysts expand(left). With sufficient intervention, cysts are reduced and kidneysreturn towards normal condition (right).

FIGS. 2A, 2B, and 2C depict the effects of time-restricted feeding in aPKD rat model. FIG. 2A depicts the cystic index (percent cystic area)for kidneys of rats on the TRF or AL diet. FIG. 2B depicts the totalnumber of cysts per kidney section in AL and TRF animals. FIG. 2Cdepicts cyst sizes of the cysts counted in 2B.

FIG. 3 depicts the abundance of pS6-positive cells in cyst-liningepithelia as a percent of the total number of DAPI-stained nuclei PKDrats on AL diet TRF diet.

FIGS. 4A, 4B, 4C, 4D, and 4E depict the effects of a ketogenic diet ondisease progression in juvenile Han:SPRD rats. FIG. 4A depicts thecystic index (percent cystic area) from NC (normal chow) and KD (ketodiet) cystic rats. [Dr6] FIG. 4B depicts serum creatinine. FIG. 4Cdepicts total number of cysts per kidney section. FIG. 4D depicts thesize of cysts counted in FIG. 4C. FIG. 4E depicts quantification ofpS6-positive cells in cyst-lining epithelia as a percent of the totalnumber of DAPI-stained nuclei in cysts. Error bars represent SD.Statistical significance determined from Mann-Whitney analysis. n=8 maleand 7 female Cy+ rats; n=13 male and 6 female wild-type rats forketogenic diet experiments (*p<0.05,****p<0.0001).

FIGS. 5A, 5B, and 5C depict PKD progression in rats treated with ketodiet. FIG. 5A depicts Cystic index (percent cystic area). FIG. 5Bdepicts the total number of cysts per kidney section. FIG. 5C depictsthe size of cysts counted in FIG. 5B.

FIGS. 6A, 6B, and 6C depict effects of fasting (acute 48-h fast) inHan:SPRD and WT rats. FIG. 6A depicts blood BHB levels in male rats.FIG. 6B depicts blood BHB levels in female rats. FIG. 6C depicts cysticindex (percent cystic area) in kidneys of fasted PKD rats.

FIG. 7 depicts the cystic index (percent cystic area) in Han:SPRD PKDrats administered BHB, vs. control rats given water or water with saltequivalent to the BHB treatment.

FIGS. 8A and 8B depict the effects of varying concentrations of BHB andcitrate, as well as BHB-citrate combination treatments, in PKD rats.FIG. 8A depicts 2-kidney to total body weight ratio in wild type (WT)and Cy+ PKD rats. FIG. 8B depicts cystic area (percent cystic area) inkidneys of the treated PKD rats.

FIG. 9 depicts PKD progression (as assessed by 2-kidney to total bodyweight ratio) in niacin-treated PKD mice that express GPR109A (Genotype1), and in GPR109A knockout PKD mice (Genotype 2).

DETAILED DESCRIPTION OF THE INVENTION

The various inventions disclosed herein are directed to dietarysupplements, pharmaceutical compositions, and other compositionscomprising BHB or BHB-related compounds, and the novel uses of suchcompositions in maintaining health, preventing disease and treatingvarious conditions. In a primary embodiment, the support of kidneyhealth and the treatment of kidney disease is encompassed by thecompositions and methods of the invention, for example, the treatment ofpolycystic kidney disease.

Part 1. Compositions of the Invention

In a first aspect, the scope of the invention encompasses compositionswhich may be used to promote wellness, prevent disease, or treat variousconditions. The compositions of the invention encompass BHB and otherketone bodies and variants thereof as well as unrelated chemicalstructures which functionally recapitulate the biological andtherapeutic effects of BHB. Therefore, the compositions of the inventionwill be referred to herein broadly as “ketonic compositions.” As setforth below, the ketonic compositions of the invention may comprise anynumber of ketone bodies, ketone body precursors, ketone body functionalmimics, and multifunctional combination products comprising two or moreketone bodies or analogs. In some implementations, the scope of theinvention encompasses multifunctional combination products comprisingketone bodies or analogs with additional functional agents that augmentor synergize the benefits of the ketones. In some embodiments, theketonic compositions of the invention comprise novel compositions ofmatter. In some embodiments, the ketonic compositions of the inventioncomprise known compositions of matter that are repurposed for noveluses.

Ketosis Mimic. In a first aspect, the ketonic composition of theinvention encompasses a ketosis mimic. As used herein, a ketosis mimicis a composition of matter which, when administered to a subject,induces or recapitulates, in whole or in part, processes or statesactive in ketosis, for example, dietary ketosis, i.e. ketosis induced byfasting or otherwise restricting glucose, for example, a blood ketonelevel of 0.5 mM or greater.

In one aspect, the ketosis mimic may comprise an exogenous ketone. Anexogenous ketone is an exogenously applied ketone body that isstructurally or functionally like BHB or other ketone bodies foundcirculating in subjects experiencing ketosis, e.g. dietary ketosis, orwhich is converted to a ketone body in vivo.

BHB and BHB Analogs. In a primary implementation, the ketosis mimic isBHB or a BHB analog. As known in the art, beta-hydroxybutyric acid,abbreviated BHB, is a ketone that is synthesized in the liver fromacetyl-CoA formed from the breakdown of fatty acids via severalintermediates and reaction steps. BHB is formed in ketosis or other lowglucose conditions. STRUCTURE 1:

For example, BHB may be employed either as the free acid or as a salt ora mixture of both. BHB or BHB salts are preferentially administered inoral formulations for gastrointestinal absorption leading todistribution via the circulation and effects of BHB in the bodyincluding the kidneys.

Likewise, analogs of BHB may also be employed in the compositions andmethods of the invention. Direct administration BHB in its free acidform may be problematic due to its acidity. Likewise, administration ofBHB salts may be problematic due to increased salt intake which may beundesired. Accordingly, in various implementations, a composition thatis converted to BHB in vivo or which mimics BHB may be used in place offree BHB or BHB salts. As used herein, “BHB analogs” broadly encompassescompositions that (1) are converted to BHB in vivo or (2) havestructural similarly to BHB and promote like physiological responses asthose promoted by BHB.

In one embodiment, the BHB analog is a precursor of BHB. A BHB precursoris a composition that is converted to BHB in vivo via one or morereactions or intermediates. In one embodiment, the BHB precursor isbutyrate. In one embodiment, the BHB precursor is beta-hydroxybeta-methylbutyrate. In one embodiment, the BHB precursor is a ketogenicamino acid or a deaminated keto-analogue of a ketogenic amino acid. Inone embodiment, the BHB precursor is acetoacetate (the physiologicalform of acetoacetic acid). In one embodiment, the BHB precursor ispoly-beta-hydroxybutyrate. In one embodiment, the BHB precursor isbeta-hydroxybutyryloxy-butyrate.

In one embodiment, the BHB precursor is 1,3 butanediol, Structure 2:

1,3 butanediol is water soluble and well tolerated by animals, includinghumans. In the body, it is efficiently converted to BHB by enzymaticand/or chemical action, for example, wherein alcohol dehydrogenasecatalyzes metabolism of 1,3-butanediol to beta-hydroxybutyraldehyde,which is subsequently oxidized to beta-hydroxybutyrate by aldehydedehydrogenase. Advantageously, 1,3 butane diol is neutral and can beadministered directly without the need to formulate as a salt. In oneembodiment, the BHB precursor is 1,3-butanediol diacetoacetate.1,3-butanediol diacetoacetate is water soluble, well tolerated inanimals and converted to BHB in vivo.

In one embodiment, the BHB analog comprises a BHB precursor comprising afat molecule which is readily converted to ketones in the body. In oneembodiment, the BHB precursor is a medium chain triglyceride (MCT), forexample, a triglyceride comprising six to 12 carbon chains. MCTs aremore efficiently converted to ketones in vivo than shorter or longerchain fats. Exemplary MCTs include caproic acid (C6), caprylic acid(C8), capric acid (C10), and lauric acid (C12).

BHB Esters. The BHB analogs of the invention further encompass esters ofBHB, and esters of BHB analogs, wherein the oxygen of the hydroxyl groupof the BHB carboxylic acid moiety (carbon 1) is conjugated with ahydroxyl group of another compound. Exemplary esters include methyl,ethyl, propyl (e.g. n-propyl or 2-propyl), butyl (e.g. tert-butyl),pentyl, hydroxylbutyl, hydroxylpropyl, glycerol, citrate, or glycolgroups joined to the 1 carbon of BHB. Other exemplary esters are3-hydroxybutyrate-(R)-1,3-butanediol monoester. Esters of BHB at the 1carbon eliminate the carboxylic acid group and associated charge. Invivo, these compositions are de-esterified by esterase enzymes,releasing BHB (or the BHB analog or mimic in some cases). Suchcompositions may have improved solubility, increased membranepermeability, improved stability, more sustained release, and increasedtolerance compared to native BHB or BHB analogs. Reference to “BHB” and“BHB analogs” herein will be understood to encompass esters of BHB andesters of BHB precursors and mimic compositions. Esters of the 3 carbonare also within the scope of the invention.

Various BHB esters are known in the art. Additional BHB esters mayinclude compositions of the formula: STRUCTURE 3

wherein R1 and/or R2 may be an alkyl moiety, for example, comprising oneto twelve carbons, for example, methyl, ethyl, propyl (e.g. n-propyl or2-propyl), butyl (e.g. tert-butyl), pentyl, hydroxylbutyl,hydroxylpropyl, glycerol, citrate, or glycol.

In some embodiments the BHB analog is a polymer of BHB or a polymer of aBHB analog. One example is polymeric BHB which is a polymeric esterbetween the carboxyl group of BHB and the hydroxyl group of BHB.Hydrolysis of polymeric BHB, for example in the gastrointestinal tract,either chemically, enzymatically or mediated by microorganisms, may leadto release of monomeric BHB and uptake into circulation.

In some embodiments, the therapeutic composition of the inventioncomprises a BHB prodrug, or BHB analog prodrug. A BHB prodrug, or BHBanalog prodrug, is a composition comprising a cleavable promoietyconjugated to BHB, or a BHB analog. In the body of a subject to whichthe prodrug is administered, the promoiety is cleaved by enzymatic orchemical action to release the BHB, or BHB analog. In a primaryembodiment, the promoiety is joined to the BHB or BHB analog by esterlinkage to the 1 and/or 3 carbon.

Various BHB analogs, including BHB esters and oligomers, are known inthe art. Any such compositions may be selected as the ketoniccomposition of the invention. In one embodiment, the BHB analogcomprises 3-hydroxybutyl 3-hydroxybutyrate, as described in U.S. Pat.No. 10,051,880, Hydroxybutyrate ester and medical uses thereof, byClarke and Veech. In one embodiment, the BHB analog comprisesR,S-1,3-butanediol acetoacetate, for example, as described in U.S. Pat.No. 9,795,590, Ketone supplements for treatment of angelman syndrome, byWeeber et al. In one embodiment, the BHB analog is an oligomer of(R)-3-hydroxybutyrate, for example, as described in U.S. Pat. No.10,559,258, Nutritional supplements and therapeutic compositionscomprising (R)-3-hydroxybutyrate derivatives, by Veech and King. The BHBanalog may comprise a composition described in U.S. Pat. No. 10,376,528,Composition comprising ketone body and nicotinamide adenine dinucleotidemodulator and methyl donor, by Schmidt or in U.S. Pat. No. 5,693,850,Nutritive water soluble glycerol esters of hydroxy butyric acid, byBirkhahn et al.

Enantiomers. BHB is a chiral composition having two enantiomers:D-β-hydroxybutyric acid (also known as R-β-hydroxybutyric acid) andL-β-hydroxybutyric acid (also known as S-β-hydroxybutyric acid). The D-and L-forms of BHB are determined by the tetrahedral orientation of thehydroxyl group (or oxy group, in BHB esters) on the third carbon of BHB.D-BHB is the most common endogenous form of BHB. L-BHB is only occurringin small amounts in cells, and thus is metabolized or cleared at aslower rate, while retaining key signaling properties of endogenousD-BHB.

Reference to “BHB” or a “BHB analog” made herein will be understood toencompass any mixture of D-BHB and L-BHB isoforms, unless the proportionof the enantiomers is specifically specified. Exemplary mixtures ofenantiomers include substantially pure formulations of D-BHB and D-BHBanalogs, for example, formulations comprising at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% D-BHB or a D-BHB analog. Likewise, the mixture ofenantiomers may comprise a substantially pure formulation of L-BHB or aL-BHB analog, for example, formulations comprising at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% L-BHB or L-BHB analog. In other embodiments,the percentage of D-BHB or D-BHB analog in the mixture may be at least5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% (racemic mixture), 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

GPR109A Activators. In one aspect, the ketonic compositions of theinvention will comprise an activator of GPR109A. GPR109A, also known ashydroxycarboxylic acid receptor 2 (HCA2), and niacin receptor 1 (NIACR1in humans) is a hydroxycarboxylic acid receptor. In humans, GPR109A isencoded by the HCAR2 gene. As used herein, a GPR109A agonist oractivator is a composition which activates, i.e. increases the activityof, GPR109A. In one embodiment, GPR109A activation is indicated by areduction in intracellular cAMP concentration. In one embodiment,GPR109A activation is indicated by a reduction in lipolysis. Activationof GPR109a occurs when a ligand or agonist binds to this receptor andcauses a conformational change that then leads to activation of anassociated heterotrimeric G-protein, in this case typically aG_(i)/G₀-type G protein. The activated G protein then regulatessubsequent intracellular signaling pathways such as the inhibition ofadenylate cyclase leading to lowering of the intracellular cAMPconcentration. Both, natural ligands—such as BHB—and artificialligands—such as certain pharmacological compounds—can act as agonists ofGPR109A.

In one embodiment, the GPR109A agonist is an exogenous ketone. Theinventors of the present disclosure have unexpectedly determined thatketones present in ketosis have GPR109A activation activity and thatthis activity underlies various beneficial processes and effects ofdietary ketosis. In one embodiment, the exogenous ketone is BHB or BHBanalog, as described above.

In alternative implementations, the GPR109A agonist is a non-ketone. Inone embodiment, the GPR109 agonist is niacin, or a niacin analog such asinositol nicotinate. It will be understood that niacin in small doses isnot sufficient to significantly activate GPR109A, and that dosage formsof less than the minimal dose, or doses administered at less than theminimal activation dose will not be considered GPR109A activators. Inone embodiment, the niacin or niacin mimic is provided (e.g. a dosageform) or administered in a biologically or therapeutically effectiveamount, being an amount sufficient to significantly activate GPR109A,for example, in various embodiments being at least 100 mg, 250 mg, atleast 500 mg, at least 750 mg, at least 1 gram, or at least 1.5 gram,for example, in human subjects. In some embodiments, niacin provided oradministered in a biologically or therapeutically effective amountcomprising a megadose suitable for activation of GPR109A, for example, adosage form or a daily administration of, for example 1 to 6 gramsdaily.

In various embodiments, the GPR109A agonist may be any of the following,as well as variants, derivatives, and polymorphs of the following. Inone embodiment, the GPR109A agonist is GSK256073, for example, asdescribed in Specher et al., 2015, Discovery and characterization ofGSK256073, a non-flushing hydroxy-carboxylic acid receptor 2 (HCA2)agonist, Eur J Pharmacol 756: 1-7. In one embodiment, the GPR109Aagonist is selected from the group consisting of(1aR,5aR)1a,3,5,5a-Tetrahydro-1H-2,3-diaza-cyclopropa[a]pentalene-4-carboxylicAcid (MK-1903), monomethylfumarate acipimox, acifran, 3-pyridine-aceticacid, 5-methylnicotinic acid, pyridazine-4-carboxylic acidpyrazine-2-carboxylic acid, LUF6281 and LUF6283, and a pyrazolylpropionyl cyclohexenamide, for example, as described in Ding et al.,2010, Discovery of pyrazolyl propionyl cyclohexenamide derivatives asfull agonists for the high affinity niacin receptor GPR109A, Bioorg MedChem Letters 21: 2721-2724; Palani et al., Discovery of SCH 900271, aPotent Nicotinic Acid Receptor Agonist for the Treatment ofDyslipidemia, ACS Med Chem Lett. 2012 3(1): 63-68. Additional GPR109agonists include compositions described in PCT International PatentApplication Publication No. WO2008051403, Acyl hydroxypyrazoles as novelagonists for high-affinity nicotinic acid receptor GPR109A, by Beresisand Colletti; U.S. Pat. No. 7,723,342, Heterocycles as nicotinic acidreceptor agonists for the treatment of dyslipidemia, by Palani et al.;United States Patent Application Publication Number US20090062269,Niacin Receptor Agonists, Compositions Containing Such Compounds andMethods of Treatment, by Raghavan et al.

In one embodiment, the GPR109A agonist is5-[3-(1-methylcyclopropyl)propyl]-1H,2H,3H,4H,7H-pyrano[2,3-d]pyrimidine-2,4,7-trione,also known as SCH900271, a nicotinic acid derivative known in the artfor the treatment of dislipideima. In one embodiment, the GPRO19 agonistis(4aR,5aR)-4,4a,5,5a-Tetrahydro-1H-cyclopropa[4,5]cyclopenta[1,2]pyrazole-3-carboxylicacid, also known as MK1903.

The present disclosure makes reference to the administration oringestion of “a ketonic composition.” It will be understood thatreference to a ketonic composition encompasses “one or more” ketonicagents, for example, one ketonic agent or a combinations of two or moredifference ketonic agents. Combinations of different ketonic agents maybe utilized, for example having different ADMET properties orphysiological effects, in order to optimize the delivery, distribution,and action of ingested or administered products or to broaden thetherapeutic effects thereof.

Synergistic Combination Products. The inventors of the presentdisclosure have advantageously determined that the efficacy of ketoniccompositions such as BHB, BHB analogs, and GPR109A agonists isdramatically, and synergistically, enhanced when the agent is combinedwith a crystal precipitation inhibiting agent. The crystal precipitationinhibiting agent, as used herein, encompasses any agent that acts toinhibit the in vivo formation of crystals such as calcium or uric acidcrystals. As is discussed below, in various contexts, crystal formationmay be pathological or may promote pathological outcomes, for example,in the kidney. By inhibiting undesirable crystal precipitation,administration of the crystal precipitation-modulating compositions ofthe invention enhances the therapeutic effects of ketonic compositionssuch as BHB, BHB analogs, and GPR109A activators. In one aspect, thescope of the invention encompasses a novel combination productcomprising a ketonic composition and a crystal precipitation inhibitor,e.g. BHB or a BHB analog (such as a BHB precursor) in combination withthe crystal precipitation inhibitor. In a related embodiment, the scopeof the invention encompasses methods of supporting health, e.g. renalhealth, or treating an enumerated condition (e.g. a renal or cysticcondition, e.g., PKD) by the administration of a biologically ortherapeutically effective amount of the combination product.

The crystal precipitation inhibitor composition may comprise any agentthat inhibits the formation of crystals in physiological contexts, forexample, the formation of calcium oxalate crystals, the formation ofcalcium phosphate crystals, and the formation of uric acid crystals. Inone embodiment, the crystal precipitation inhibitor is a calciumchelator. In some embodiments, the crystal precipitation inhibitorcomposition is a buffering agent or pH-modulating agent that changes pHto inhibit crystal precipitation, for example, a pH-raising agent whichinhibits uric acid crystal formation.

In a primary embodiment, the crystal precipitation-modulatingcomposition is citrate. Citrate, as referred to herein, encompassescitric acid itself, or salts and esters of citric acid. For example,citrate may comprise a mono-, di- or tri-valent salt of citrate withsodium, potassium, magnesium, calcium, other cations, or mixtures ofcations, or mixtures of citric acid with citrate salts. For example,citrate may comprise a mono-salt, for example a mono-sodium salt, forexample, the sodium salt of STRUCTURE 4, or other salts wherein adifferent cation is used:

In one aspect, the scope of the invention encompasses a novelcombination product comprising a ketonic composition and citrate, e.g.BHB or a BHB analog (such as a BHB precursor) in combination withcitrate. In a related embodiment, the scope of the invention encompassesmethods of supporting health, e.g. renal health, or treating anenumerated condition (e.g. a renal or cystic condition, e.g., PKD) bythe administration of a biologically or therapeutically effective amountof the combination product comprising BHB or a BHB analog an citrate.Citrate may be administered as a salt of magnesium, potassium, calcium,sodium, or other forms. The use of citrate alone in the treatment ofpolycystic kidney disease in rats has been disclosed, for example, byTanner and Tanner, Dietary Citrate Treatment of Polycystic KidneyDisease in Rats Nephron Physiol 2003; 93:p 14-p 20 and in Tanner,Tanner, Potassium citrate/citric acid intake improves renal function inrats with polycystic kidney disease, JASN July 1998, 9 (7) 1242-1248.However, the combination of BHB or other ketones and citrate has notbeen disclosed.

In some embodiments, the crystal precipitation-modulating composition isa citrate analog, for example, a composition having structuralsimilarity to citrate and retaining the crystal precipitation-modulatingproperties of citrate. Exemplary citrate analogs include derivatives ofcitric acid known in the art. Exemplary citrate analogs include estersof citric acid (e.g. monoester, diester, triester), amides of citricacid, 1,3 dioxal-4-ones, sidophores, peroxycitric acid, hydroxycitrate,and tricarballylic acid. Exemplary citrate analogs include compositionsdescribed in Milewska, 1988, Citric Acid, its natural and syntheticderivatives, Z Chem 28: 204-211.

In some embodiments, the crystal precipitation inhibitor is acomposition that alkanizes urine, such as a carbonate or bicarbonatecomposition, such as magnesium carbonate or bicarbonate, calciumcarbonate or bicarbonate, or sodium carbonate or bicarbonate. In someembodiments, the crystal precipitation-modulating composition is acompound that can decrease the level of uric acid in the urine and maybe used to treat hyperuricemia. Such compounds will decrease the risk offormation or uric acid crystals. Three examples are allopurinol,oxypurinol and febuxostat, inhibitors of xanthine oxidase, that act onpurine catabolism, reducing the production of uric acid. Another exampleis Lesinurad which inhibits the function of transporter proteinsinvolved in renal uric acid reabsorption (uric acid transporter 1[URAT1] and organic anion transporter 4 [OAT4]), and lowers serum uricacid levels. Another example is Rasburicase, a recombinant urate-oxidaseenzyme, which converts uric acid to allantoin (an inactive and solublemetabolite of uric acid).

In various implementations, the therapeutic compositions of theinvention comprise a combination of a ketonic composition, e.g., one ormore of BHB, a BHB analog, and/or a GPR109A activator, in combinationwith one or more crystal precipitation-modulating compositions, forexample, a combination of BHB and citrate, for example an admixture ofthe two in a capsule, tablet, powder, beverage, or edible item. Byformulating the therapeutic compositions of the invention as combinationproducts, the recipient may advantageously be administered a singleagent with dual, synergistic therapeutic properties.

In an alternative implementation, the combination product is providedwith the ketonic composition and crystal precipitation inhibitor as twoseparate dosage forms, optionally packaged in a single package. Forexample, two separate tablets, capsules, powders, e.g. a BHB capsule anda citrate capsule, may be provided with instructions to take them at thesame time. By formulating the therapeutic compositions of the inventionas individual entities, the recipient may benefit from the synergistictherapeutic properties but advantageously has the option to modify theindividual doses according to specific needs.

It will be understood that citrate is a common additive to dietarysupplements and other food items. For example, sodium citrate orpotassium citrate are known additives used as flavoring agents,preservatives, and pH buffers. The quantity of citrate used for suchpurposes is therapeutically insignificant. The scope of the inventionencompasses synergistic combination products comprising citrate whereinthe quantity of citrate is biologically effective, i.e. sufficient toinhibit crystal precipitation in vivo, for example, crystalprecipitation in the kidney. For example, when citrate is configured fororal administration, the biologically effective dosage may be in therange of 100 mg to 10 grams citrate per day. In various embodiments, thecitrate dose is about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1,000 mgcitrate or citrate salt per dose, per dosage form, or cumulative dosingper day. In an alternative method of assessing citrate dose, the dosemay be in the range of 0.1 mmol-10 mmol citrate/100 g body weight perdosage, or per day, for example, a dosage of about or at least 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2. 2.3, 2.4, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 mmol citrate/100 gbody weight per dose, or cumulative dose per day. In another method ofdosage calculation, the citrate dosage is in the range of 0.1 to 10 mgcitrate or citrate salt per m² body area per dosage or per day, forexample at being about or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5., 6.0, 6.5,7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10 mg per m² body area.

In some implementations, the biologically effective citrate is definedin terms of its abundance relative to the co-administered ketonic agent,e.g. BHB or a BHB analog. For example, in various embodiments, the ratioof citrate molecules, or mass of citrate, to BHB or BHB analog molecules(or mass) is in the range of 1:10 to 10:1. Exemplary ratios of citrateto BHB or BHB analog (by molecule or by mass) are in the range of 1:5,1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 or 5:1.

In some implementations, the amount of citrate in the combinationproduct is described in terms of the weight percentage of citrate in thecombination product. In one embodiment, the weight percentage of citrateis calculated as proportional mass of citrate to the mass of citrate andthe one or more ketonic compositions (e.g. the weight percentage ofcitrate with respect to active ingredients only). With respect to thismeasure, the weight percentage of citrate may be in the range of atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 70%, at least 80%, at least 90% or atleast 95%. In one embodiment, the weight percentage of citrate iscalculated as the proportional mass of citrate to the mass of the entireformulation, including citrate, ketonic compositions, and carriers andother inert ingredients (e.g. the weight percentage of citrate withrespect to the entire product). With respect to this measure, the weightpercentage of citrate may be in the range of at least 5%, at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 70%, at least 80%, at least 90% or at least 95%.

Bi-Functional Molecules. In one embodiment, the scope of the inventionencompasses novel compositions, advantageously comprising abi-functional molecule comprising one or more ketonic compositions, e.g.one or more of BHB, a BHB analog, or a GPR109A agonist, conjugated toone or more molecules of a crystal precipitation-modulating agent. Invarious embodiments, the dietary supplements and therapeuticcompositions of the invention comprise such compositions. The use ofsuch compositions advantageously provides a ketosis mimic and/or GPR109Aagonist in combination with a crystal precipitation inhibitor in asingle composition, at a desirable stoichiometric ratio. Additionally,the bi-functional molecules may be designed to be neutral or lessacidic, thereby avoiding or reducing the need for salt formulation.

In one embodiment, the bi-functional molecule comprises a BHB-citrateester, comprising one or more BHB molecules and/or one or more a BHBanalog molecules conjugated to citrate. Exemplary BHB-citrate estersinclude STRUCTURE 5:

wherein R1, R2, or R3 may be hydrogen, alkyl, or another composition,and at least one of R1, R2, and R3 is ketonic composition. In oneembodiment, R1, R2, or R3 may be hydrogen, alkyl, or anothercomposition, and at least one of R1, R2, and R3 is BHB. In oneembodiment, R1, R2, or R3 may be hydrogen, alkyl, or anothercomposition, and at least one of R1, R2, and R3 is a BHB analog. In oneembodiment, R1, R2, or R3 may be hydrogen, alkyl, or anothercomposition, and at least one of R1, R2, and R3 is 1,3 butanediol. Inone embodiment, R1, R2, or R3 may be hydrogen, alkyl, or anothercomposition, and at least one of R1, R2, and R3 is a GPR109A activator.

Exemplary bi-functional molecules include Structure 6:

Structure 7:

and

Structure 8:

Synthesis of bi-functional molecules. The bi-functional BHB-citrateesters and like composition of the invention may be synthesizedaccording to known methods for the esterification of citrate and likemolecules. For example Fischer-Speier esterification may be employed byrefluxing citric acid with an excess of 1,3-butanediol in the presenceof an acid catalyst such as sulfuric acid. Such a reaction is expectedto yield a mixture of structures 7 and 8 as well as mixed esters withthe 1,3-butanediol component esterified variously via the 1 and 3positions in addition to other possible combinations. In all such cases,the in vivo hydrolysis of the bi-functional esters is expected to yieldthe functional components citrate and 1,3-butanediol. If more specificand pure bi-functional esters are desired, many commonly known chemicalsynthesis steps are available. For example, the hydroxyl group of citricacid could be protected by acetylation, e.g. using acetic anhydride,prior to carrying out the Fischer-Speier esterification described above.Similarly, precursors to 1,3-butanediol—in which either the 1-OH groupor the 3-OH group is protected—could be used as starting material in theFischer-Speier esterification.

Combination Product Comprising Lipotoxic Compositions. Due to themetabolic inflexibility of epithelial cells in cysts, such as PKD cysts,for example, ADPKD cysts, in some implementations, the compositions andmethods of the invention encompasses a combination product comprising aketonic composition, e.g., one or more of BHB, a BHB analog, and/or aGPR109A activator, and a lipotoxic agent. The lipotoxic agent maycomprise any fatty acid or other composition which create toxicity forcells that are dependent upon glucose for energy production and may bedefective in beta-oxidation of fatty acids, i.e. having enhancedsensitivity to lipotoxicity from lipids that accumulate but which cannotbe metabolized. Exemplary glucose dependent cells include cystepithelial cells, including PKD cyst epithelial cells and liver cystepithelial cells, and cancer cells.

In one embodiment, the lipotoxic composition is an MCT, as describedabove. Exemplary MCTs include triglycerides comprising six to 12-carbonchains, including caproic acid, caprylic acid, capric acid, and lauricacid.

Additionally, unsaturated fatty acids may act as lipotoxic agents forglucose-dependent cells. Exemplary unsaturated fatty acids includemonounsaturated and polyunsaturated fats such as a-Linolenic acid,Stearidonic acid, Linoleic acid, Linolelaidic acid, γ-Linolenic acid,Palmitoleic acid, Vaccenic acid, Oleic acid. Such unsaturated fattyacids may be administered alone or in mixtures as free fatty acids ortheir salts or as triglycerides. Alternatively, these unsaturated fattyacids may be administered in the form of natural fats and oils that arerich in these unsaturated fatty acids, for example including oils fromflaxseed, walnuts, chia, hemp, and other vegetable oils.

The lipotoxic agents advantageously compositions that can be takenorally and which are used in various food products and supplements. Inone embodiment, the lipotoxic agents are included in dietary supplementsof the invention. The dosage of lipotoxic agents will be in the range of0.5-100 g per day, for example about 1, 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, or 90 g per day.

In certain embodiments, the combination product comprising a ketoniccomposition and a lipotoxic composition further includes a crystalprecipitation-modulating agent (e.g. citrate). For example, thecombination product may comprise BHB (or a BHB analog) in combinationwith citrate (or a citrate analog) in combination with one or morelipotoxic compositions, representing a tri-functional agent.

Formulations of the Ketonic Compositions and Combination Products. Itwill be understood that the ketonic compositions and combinationproducts containing such described herein may be formulated in variousways for effective and controlled delivery. It will be understood thatthe compositions may comprise the active agents described herein incombination with any number of ingredients that facilitate effectiveingestion, administration, delivery, adsorption, distribution, andmetabolism of the active agents. In various embodiments, thecompositions described herein comprise dietary supplements, ortherapeutic compositions (including therapeutic compositions formulatedas dietary supplements). Further, all compositions described hereincontaining or comprising ketonic agents may be considered “medicaments.”Furthermore, the act of manufacturing, including formulating, ketonicagents into dietary supplements, therapeutic compositions, or anyingested or otherwise administered compositions will be presumed to beperformed under any specified processes therefor, and if no process isspecified, then the act of manufacture, including formulation, will beassumed to be achieved by standard means known in the art. Thus recitalof any described compositions wherein ketonic compositions are included,will be presumed to encompass an implied method of making suchmedicaments.

In various embodiments, the ketonic composition (e.g. BHB, BHB analog,and/or GPR109A activator) is formulated as a salt. Pharmaceutical saltformulations are known in the art and may impart higher solubility,stability, and bioavailability to the therapeutic molecules. Exemplarysalts include salts of amino acids, alkali metals, alkaline earthmetals, transition metals, for example: arginine salts, calcium salts,chromium salts, citrulline salts, cobalt salts, copper salts, creatinesalts, glutamine salts, histidine salts, iron salts, isoleucine salts,leucine salts, lithium salts, lysine salts, magnesium salts, manganesesalts, molybdenum salts, ornithine salts, potassium salts, seleniumsalts, sodium salts, and zinc salts.

In one primary implementation, the ketonic agents of the invention areformulated in a low-salt or zero-salt formulation, to avoid stressingthe body, especially the kidneys, of the recipient. For example, alow-sodium and low-potassium and low-calcium formulation may comprise aformulation that would lead to an added sodium or potassium or calciumintake of no more than 500 mg/day each, preferably less.

The ketonic compositions of the invention may be formulated forefficient delivery by a selected route. The ketonic compositions of theinvention may be formulated in combination with pharmaceuticallyacceptable excipients, carriers, diluents, release formulations andother drug delivery or drug targeting vehicles, as known in the art.

Advantageously, the ketonic compositions of the invention are amenableto efficient oral delivery and may be formulated therefor. Oralformulations may include any number of fillers, flavoring agents,coloring agents, sweeteners, and other compositions used in the art fororal delivery of agents. In one embodiment, the pharmaceuticalcomposition is formulated for oral delivery in tablets or capsules. Inone embodiment, the pharmaceutical composition is formulated as a powderto be mixed with liquid. In one embodiment, the ketonic composition isformulated as a liquid containing the therapeutic agent, i.e. solutions,such as water or other flavored drinks containing BHB or a BHB analog.

In alternative implementations, the ketonic compositions of theinvention are formulated for administration via non-oral routes,including, for example, intravenous, intra-arterial, intraperitoneal,intrapulmonary, oral, inhalation, intravesicular, intramuscular,intra-tracheal, subcutaneous, intraocular, intrathecal, transmucosal,and transdermal delivery. In one embodiment, the ketonic compositions ofthe invention may be formulated for microbubble delivery for example,ultrasound meditated microbubble delivery, for site-specific delivery.In one embodiment, the ketonic compositions comprises nanoparticlescontaining or functionalized with the selected active agent of theketonic composition, for delivery by nanoparticle-based deliverymethods. In one embodiment, the ketonic composition comprises theselected therapeutic agent admixed with a polymeric material for timedrelease elution of the agent or to prevent premature digestion of thematerial in the digestive tract. In one embodiment, the ketoniccomposition comprises chemically conjugated magnetizable particles oruse in magnetic-directed drug delivery methods, as known in the art. Inone embodiment, the ketonic composition of the invention is coated ontoan implant or drug eluting device.

In one embodiment, the therapeutic composition of the inventioncomprises a targeting moiety that facilitates delivery to a target celltype or organ, such as the kidneys, liver, cysts, or other sites. In oneembodiment, the ketonic composition of the invention is conjugated to apolymeric immunoglobulin such as a polymeric IgM or IgA. Such agents maybe efficiently targeted to renal cysts, for example, cysts present inthe case of polycystic kidney disease.

Administration. The various methods of the invention may encompass theadministration of an agent to a subject. Administration, as used herein,may encompass the act of providing the agent to the subject or actuallyintroducing the agent into the subject. Administration as used hereinwill encompass self-administration. For example, in a primaryimplementation of the methods of the invention, dietary supplements areingested by a subject. Such ingestion will be considered administration,i.e., self-administration of the enumerated composition. Administrationmay also comprise provision of a selected composition to the subject,for example, providing food or liquids that contain the composition toan animal.

Dietary Supplements. In a primary implementation of the methods of theinvention, the ketonic composition is formulated and administered as adietary supplement. A dietary supplement is a composition that isadministered orally, for example, in food or drink, with food or drink,or which otherwise supplements the regular intake of food. From aregulatory standpoint, a dietary supplement is generally a compositionthat can be purchased and used without a prescription. For example, inthe United States, per the Food and Drug Administration and The DietarySupplement Health and Education Act of 1994, a dietary supplement is “aproduct intended to supplement the diet that bears or contains one ormore of the following dietary ingredients: a vitamin, a mineral, an herbor other botanical, an amino acid, a dietary substance for use by man tosupplement the diet by increasing the total dietary intake, or aconcentrate, metabolite, constituent, extract, or combination of anyingredient just described.”

The dietary supplements of the invention may encompass any formulationfor oral delivery. In a primary implementation, the ketonic compositionsof the invention are ingested in liquids, for example, being formulatedin any number of beverage formats, including infused water, sportsdrinks, energy drinks, teas, fruit juices, or meal replacementbeverages. In one embodiment, the ketonic compositions of the inventionare formulated as a powder which may be dissolved in water, or otherdrinks such as fruit juice, coffee, or tea. In some embodiments, thepowder is packaged in individual packets or vials at a measured dose. Insome embodiments, the powder is packaged with a scoop which measures thecorrect dose for mixing with liquid and instructions for doing so at aspecified frequency.

In an alternative dosage form, the dietary supplement of the inventionis formulated as a capsule, tablet, or chewable tablet. In someimplementations, the dietary supplements of the invention are ingestedas a component of an edible item, for example, being incorporated intogummies, cookies, snack bars, meal replacement bars and other bars,cereals, biscuits, crackers, confectionery items, and probioticformulations including yogurt, and other food items. In some cases thedietary supplements of the invention are formulated as products fornon-human animals, such as chow, feed, treats or snacks, or supplementedpet food.

In the case of combination products comprising a ketonic composition incombination with a secondary agent (e.g. a crystal precipitationinhibitor such as citrate or a lipotoxic agent such as unsaturated fattyacids), the dietary supplements are preferably formulated as a singleproduct containing both the ketonic composition (e.g. BHB or BHB analog)and the secondary agent so that only a single product is required to bedigested. However, it will be understood the scope of the inventionincludes separate formulations and dosage forms of the ketoniccomposition and secondary agents, to be ingested separately.

Part 2: Methods of Use

The scope of the invention encompasses numerous methods directed to theuse of ketonic compositions for the maintenance of health, prevention ofdisease, and in therapeutic applications. In one aspect, the scope ofthe invention encompasses methods of using dietary supplementscomprising ketonic compositions for supporting health. In anotheraspect, the scope of the invention encompasses the use of ketoniccompositions in therapeutic methods for the prevention and treatment ofvarious diseases and pathological conditions.

The methods disclosed herein will be directed to the use of ketoniccompositions by, and administration of ketonic compositions to,subjects. The subject may be a human subject, for example, in somecontexts, a patient. The subject may comprise a non-human animal of anyspecies, including test animals, veterinary subjects, pets, andlivestock, for example, any of mice, rats, dogs, cats, sheep, goats,cows, pigs, horses, camels, non-human primates, or other animals. Forexample, polycystic kidney disease is prevalent in dogs and cats.

In certain embodiments, the subject of the method will be a subject inneed of treatment for a selected condition. For example, the subject maybe a subject suffering from a condition, may be symptomatic of aselected condition, or may be at risk of a selected condition.

The methods of the invention encompass the ingestion and administrationof ketonic agents in a biologically or therapeutically effective amount.In one measure, a biologically or therapeutically effective amount is anamount of ketonic agent ingested as dietary supplement or administeredtherapeutically that is sufficient to induce a ketonic response. Such aresponse may be indicated by assaying the elevated blood concentrationof a ketone body such as BHB. A response could also be measured byassaying a change in the blood levels of fatty acids that can be used asan indicator of the anti-lipolytic consequences of GPR109a activation.In one measure, a ketonic response is the elevation of ketones in theblood or other compartment of the subject, for example, any significantelevation compared to subjects not experiencing ketosis. In one measure,a ketonic response is the increase in GPR109A activation in selectedtarget cells of the subject, for example, any significant increase inGPR109A activity compared to subjects not ingesting or administered aketonic agent. In one measure, a ketonic response is the attainment of aspecific physiological outcome or state, for example, the mean or mediandosage required to attain the selected outcome in a significant portionof representative subjects, compared to subjects not ingesting oradministered the ketonic agent. Selected outcomes may encompass anyrelevant outcome, such as reduced symptoms of a selected condition, forexample, reduced size or abundance of cysts in the case of cysticdisease, improved organ or cellular function, and other physiological orhealth measures.

The dietary supplements and therapeutic ketonic agents of the inventionare ingested or administered in a dose, being an amount of ketoniccomposition ingested or otherwise received at a time or over a specifiedtimeframe (e.g. a daily dose). Doses may be referred to as approximateamounts, for example, being “about X mg.” “About,” as used herein means,in various embodiments, within plus-or-minus 10%, plus-or-minus 5%, orplus-or-minus 1% of the enumerated value.

In various embodiments, the ingested or administered ketonic compositionis BHB, or a BHB analog, e.g. a BHB precursor, configured for oraladministration, wherein the administration is oral, and the biologicallyeffective or therapeutically effective dosage is in the range of 0.1 to50 grams BHB per day, e.g., for humans. For example, daily doses ofabout or at least 100 mg, about or at least 250 mg, about or at least500 mg, about or at least 1 gram, about or at least 2 grams, about or atleast 3 grams, about or at least 4 grams, about or at least 5 grams,about or at least 6 grams, about or at least 7 grams, about or at least8 grams, about or at least 9 grams, about or at least 10 grams, about orat least 12 grams, about or at least 15 grams, or about or at least 20grams may be ingested or administered. In some embodiments, the dosageof BHB, BHB analog, including BHB precursors, is in the range of 10-200mg/Kg body weight, for example, 10, 25, 50, 75, 100, 125, 150, 175, or200 mg./Kg. For non-human animal subjects, such as animal models orpets, the exemplary dosages above may be adjusted based on thedifference in body weight or body surface area. In one embodiment, thenon-human is a small animal (e.g. rodent, dog, or cat) and thebiologically or therapeutically effective amount of BHB or BHB analog isin the range of 10-150 mg/Kg, in various embodiments, for example, beingabout 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 100, 120, 150 mg/Kg.

In an alternative measure, the biologically effective or therapeuticallyeffective amount of BHB or BHB analog is determined as the amountnecessary to raise blood ketone levels (e.g., BHB levels) to a selectedthreshold indicative of ketosis, for example, a dose sufficient toattain a blood concentration of BHB at least 0.250 to 5 mMol BHB perliter of blood, for example, about or at least 0.250, about or at least0.500, about or at least 0.750, about or at least 1.0, about or at least2.0, about or at least 3.0 mMol BHB per liter of blood.

In the case of GPR109A activators, the biologically or therapeuticallyeffective dose will be determined by the potency, ADMET, and deliveryroute of the selected GPR109A activator. This class of compositionsincludes potent small molecules that act in the picomolar to nanomolarrange, as well as ketones and other orally available compositions (e.g.niacin) that require higher concentrations to induce GRP109A activationin selected target cells (e.g. cyst epithelial cells). Accordingly, thebiologically or therapeutically effective dose of GPR109A activator maybe determined by one of skill in the art based on the properties of theagent and the desired therapeutic outcome.

In the case of a combination product comprising a crystal precipitationinhibitor, e.g. citrate, a biologically or therapeutically effectiveamount is an amount of crystal precipitation inhibitor ingested oradministered that is sufficient to inhibit crystal formation in vivo,e.g. uric acid crystal formation or calcium crystal formation, orsufficient to induce conditions that inhibit crystal formation, such asincreased urinary concentration of citrate to antagonize calcium crystalprecipitation, or increased urinary pH to antagonize uric acid crystalprecipitation, or reduced blood or urine levels of uric acid toantagonize uric acid crystal precipitation.

In various embodiments, the selected crystal precipitation inhibitor,for example, citrate, is configured for oral administration, theadministration is oral, and, for example in humans, the biologically ortherapeutically effective dose ingested or administered may be in therange of 100 mg to 15 grams, for example, at least or about 100 mg, atleast or about 200 mg, at least or about 300 mg, at least or about 500mg, at least or about 1 gram, at least or about 2 grams, at least orabout 3 grams, at least or about 4 grams, at least or about 5 grams, atleast or about 6 grams, at least or about 7 grams, at least or about 8grams, at least or about 9, grams, or at least or about 10 grams. Insome embodiments, the dosage of citrate or citrate analog is in therange of 5-100 mg/Kg body weight, for example, in various embodiments,being 5, 10, 20, 25, 30, 40, 50, 55, 65, 70, 75, 80, 85 90, or 100mg/Kg. For non-human animal subjects, such as animal models or pets, theexemplary dosages above may be adjusted based on the difference in bodyweight or body surface area. In one embodiment, the non-human is a smallanimal (e.g. rodent, dog, or cat) and the biologically ortherapeutically effective amount of citrate or citrate analog is in therange of 10-100, for example, 25-75 mg/Kg, in various embodiments, forexample, being about 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 80, 90 or100 mg/Kg.

The dosage of the crystal precipitation inhibitor, e.g., citrate, in thedietary supplement may be determined by alternate measures. For example,in one embodiment, the crystal precipitation inhibitor, such as citrate,is ingested or administered at a dosage of between 0.1 mmol-10 mmol/100g body weight per day, for example, at least 0.5, at least 1, at least2, at least 5, at least 7.5 or at least 10 mmol/100 g body weight perday. In another embodiment, the crystal precipitation inhibitor, such ascitrate, is ingested or administered at a dosage of between 0.5 to 10mg/citrate or citrate salt per m² body area per day, for example atleast 1, at least 2, at least 3, at least 4, or at least 5 mg per m²body area, per day.

Doses may be ingested or administered according to a selected health ortreatment regimen. In some embodiments, doses are ingested oradministered for a selected period of time, e.g. a period of days,weeks, or months. In some implementations, especially in preventativemethods, chronic, long-term ingestion or administration is contemplatedfor maintaining beneficial, therapeutic, or preventative effects. In aprimary embodiment, the administration is daily with one or more dosagesingested or administered per day.

Multiple such dosages may be administered during the day, to arrive acumulative dosage near, at, or above the biologically effective ortherapeutically effective daily dosage, for example, two, three, four,or more doses per day. Dosage forms (e.g. capsules, packets ofdissolvable powder for drinks, scoops for measuring such powder, etc.)may be configured for fractions of the daily dose. In someimplementations, two or more dosages are delivered per day, for example,a morning and an evening dose, a dosage administered before or aftereach meal, a dosage delivered every four hours, six hours, twelve hours,or at other selected time intervals. Advantageously, a low dosage ofBHB, administered multiple times per day, may be more palatable than afull daily dosage, wherein the unpleasant taste of BHB is an issue.Likewise, by administering low dosages of citrate over multiple dailyadministrations, gastrointestinal issues associated with long termcitrate consumption can be alleviated. In alternative embodiments, thedietary supplement is consumed at less than daily frequency, forexample, once a week, twice a week, three times a week, etc.

Exemplary Dietary Supplements and Therapeutic Compositions. The methodsof the invention will encompass the administration or ingestion ofketonic compositions in a dietary supplement or a therapeuticcomposition. The dietary supplement or therapeutic composition maycomprise any of the ketonic compositions, combination products, andformulations, as disclosed in the previous sections. In variousembodiments, the dietary supplement or a therapeutic compositioncomprises an exemplary formulation as forth below.

The dietary supplement or a therapeutic composition will comprise aketonic composition. The ketonic composition may comprise one or more ofBHB, a BHB analog, a GPR109A agonist, or a combination of the foregoing.The formulations may comprise a combination of two or more differentketonic compositions. In some embodiments, ketonic composition comprisesa BHB analog, wherein the BHB analog may comprises an ester of BHB, aBHB precursor, 1,3-butanediol, or a bi-functional composition comprisingone or more molecules of BHB, a BHB analog, and/or a GPR109A agonistconjugated to one or more molecules of citrate. In some embodiments, thebi-functional molecule is Structure 5, Structure 6, Structure 7, orStructure 8. In some embodiments, the composition comprises L-BHB, forexample, at least 10% L-BHB, about 50% L-BHB, or substantially pureL-BHB. In some embodiments, the ketonic composition comprises a GPR109Aagonist, wherein the GPR109A agonist comprises a composition selectedfrom the group consisting of niacin, a niacin analog, clotrimazole,GSK256073, MK-1903, monomethylfumarate acipimox, acifran,3-pyridine-acetic acid, 5-methylnicotinic acid, pyridazine-4-carboxylicacid pyrazine-2-carboxylic acid, LUF6281, LUF6283, a pyrazolyl propionylcyclohexenamide, an acyl hydroxypyrazole, and SCH900271.

The dietary supplement or therapeutic composition will comprise aketonic composition in a biologically or therapeutically effectiveamount. In some embodiments, the biologically or therapeuticallyeffective amount is an amount sufficient to attain blood ketone levelsat a level indicative of ketosis, for example, a dose sufficient toattain a blood concentration of BHB at least 0.250 to 5 mMol BHB perliter of blood, for example, about or at least 0.250, about or at least0.500, about or at least 0.750, about or at least 1.0 mMol BHB perliter. In various embodiments, the ketonic agent comprises BHB and thebiologically or therapeutically effective amount is an amount in therange of 0.1 to 50 grams BHB, for example, 0.1 to 50 grams BHB per day.In various embodiments the biologically or therapeutically effectiveamount is about or at least 100 mg, about or at least 250 mg, about orat least 500 mg, about or at least 1 gram, about or at least 2 grams,about or at least 5 grams, or, about or at least 10 grams.

In various embodiments the dietary supplement or therapeutic compositionwill comprise a ketonic composition in combination with a biologicallyor therapeutically effective amount of a crystal precipitationinhibitor. In one embodiment, the biologically or therapeuticallyeffective amount of a crystal precipitation inhibitor is an amountsufficient to inhibit in vivo crystal precipitation, for example,sufficient to inhibit the formation of calcium oxalate crystals, theformation of calcium phosphate crystals, and/or the formation of uricacid crystals. In some embodiments, the crystal precipitation inhibitoris a calcium chelator. In some embodiments, the crystal precipitationinhibitor is a buffering agent or pH-modulating or alkanizing agent.

In various embodiments the crystal precipitation inhibitor is selectedfrom the group consisting of citrate, hydroxycitrate, esters of citricacid (e.g. monoester, diester, triester), amides of citric acid, 1,3dioxal-4-ones, sidophores, peroxycitric acid, hydroxycitrate, andtricarballylic acid, a composition that alkanizes urine, a carbonate orbicarbonate, magnesium carbonate or bicarbonate, calcium carbonate orbicarbonate, or sodium carbonate or bicarbonate, allopurinol, oxypurinoland febuxostat, inhibitors of xanthine oxidase, Lesinurad, Rasburicase,and a calcium chelator. In one embodiment the crystal precipitationinhibitor is citrate. In various embodiments, the biologically effectivebiologically or therapeutically effective amount is at least 100 mgcitrate, for example, 200 mg citrate per day, at least 500 mg citrate,for example, 500 mg citrate per day, citrate at a weight percentage inthe dietary supplement or therapeutic composition of at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

In various embodiments the dietary supplement or therapeutic compositionwill comprise a ketonic composition in combination with a biologicallyor therapeutically effective amount of a lipotoxic agent. In oneembodiment, the biologically or therapeutically effective amount of alipotoxic agent is an amount sufficient to cause lipotoxic effects inglucose-dependent cells, e.g., PKD cyst epithelial cells. in variousembodiments, biologically or therapeutically effective amount of alipotoxic agent is in the range of 0.5-100 g per day, for example about1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 90g per day. In one embodiment, the lipotoxic agent comprises MCT. In oneembodiment, the lipotoxic agent comprises fatty acids.

In one embodiment, the dietary supplement or therapeutic compositioncomprises a ketonic composition in combination with both a biologicallyor therapeutically effective amount of a crystal precipitation inhibitorand a biologically or therapeutically effective amount of a lipotoxicagent.

The dietary supplements of the invention are by definition formulatedfor oral delivery. In various embodiments, the therapeutic compositionsmay also be formulated for oral delivery. In one embodiment, thetherapeutic composition is formulated for oral delivery as a dietarysupplement. In various embodiments the dietary supplement or therapeuticcomposition is formulated as a capsule, a chewable tablet, a tablet, adissolvable powder, a beverage, or a food item.

In the case of dietary supplements or therapeutic compositionscomprising a ketonic composition in combination with a secondary agent(e.g. a crystal precipitation inhibitor such as citrate or a lipotoxicagent such as unsaturated fatty acids), the dietary supplement ortherapeutic composition may be formulated as a single product containingboth the ketonic composition (e.g. BHB or BHB analog) and the secondaryagent so that only a single product is required to be ingested oradministered. In another embodiment, the ketonic composition and thesecondary agent(s) (e.g. a crystal precipitation inhibitor such ascitrate or a lipotoxic agent) are provided together in separate dosageforms, for example, capsules or chewable tablets, for example, to beingested at the same time.

Methods of Supporting Health by the Use of Dietary Supplements. In oneaspect, the scope of the invention encompasses novel methods ofpromoting health and wellness by the use of dietary supplementscomprising ketonic compositions. In various embodiments, the function ofthe compositions and methods of the invention will be described as“supporting health,” for example, “supporting renal health.” As usedherein, supporting a enumerated type of health will encompass anyactivity that maintains, enhances, improves, or promotes relevant organor cellular function, for example, renal function in the case ofsupporting renal health. In various embodiments, the support of healthis achieved by ameliorating the risk, progression, and/or severity of acondition. Ameliorating the risk, progression, and/or severity of acondition may encompass any number of physiological, health, or medicalprocesses, such as treating or preventing the condition in any way,reducing, reversing, or curing symptoms, pathologies, or pathologicalprocesses of the condition, slowing or halting the progression of thecondition, or any other therapeutic or beneficial effect.

Methods of Treatment. Certain methods of the invention encompass theprevention or treatment of a selected condition. As used herein,treatment will encompass any number of therapeutic effects and outcomeswith respect to a selected condition, including, for example: areduction in the severity of symptoms of the condition; the inhibitionof pathological processes underlying the condition; the reversal ofpathological events or processes of the condition; halting or slowingthe progression of the condition; or a reduction in morbidity and/ormortality associated with the condition. Treatment, as used herein, willfurther encompass prevention of an enumerated condition. As used herein,prevention will encompass any number of actions with respect to aselected condition, for example: preventing the onset of the condition;reducing the probability of the condition occurring; halting the furtherprogression of the condition, ameliorating underlying physiologicalparameters that promote the condition, or any other preventative action.As used herein, treatment will further encompass enhancements of targetcell or organ function, such as quantitatively or qualitatively improvedfunction, for example, in certain implementations, improved function,restoring normal function, or maintaining function.

Methods of Supporting Renal Health and Treating Renal Conditions. In aprimary aspect, the scope of the invention encompasses the use ofketonic compositions in dietary supplements and therapeutic compositionsto address various aspects of renal health and renal conditions.

In one implementation, the scope of the invention encompasses a dietarysupplement comprising a ketonic composition for use in a method ofsupporting renal health in a subject. In a related embodiment, the scopeof the invention encompasses the use of a ketonic composition in amethod of making a medicament for the support of renal health. In arelated implementation, the scope of the invention encompasses a methodof supporting renal health in a subject by the administration to thesubject (for example, including self-administration, i.e. ingestion) ofa biologically effective amount of a dietary supplement comprising aketonic composition. In various embodiments, the dietary supplementcomprises a ketonic composition in combination with a crystalprecipitation inhibitor and/or a lipotoxic agent. In one embodiment,supporting renal health means maintaining or enhancing one or moremeasures of renal function. For example, renal function may be assessedby glomerular filtration rate, creatine clearance rate, urine output,proteinuria, or other measures of renal function known in the art. Inone embodiment, supporting renal health in a subject is achieved byameliorating the subject's risk, progression, and/or severity of one ormore renal conditions.

In various embodiments, the scope of the invention further encompassesthe use of therapeutic compositions comprising ketonic compositions forthe treatment of renal conditions. In one embodiment, the scope of theinvention encompasses a ketonic composition for use in a method oftreating a renal condition. In a related embodiment, the scope of theinvention encompasses the use of a ketonic composition in a method ofmaking a medicament for the treatment of a renal condition. In oneembodiment, the scope of the invention encompasses a method of treatinga renal condition in a subject in need of treatment therefor byadministering to the subject a therapeutically effective amount of aketonic composition. The ketonic composition may be delivered orprovided in a therapeutic composition, for example, a formulationcomprising other components and/or active ingredients. In someembodiments, the dietary supplement comprises a ketonic composition incombination with a crystal precipitation inhibitor and/or a lipotoxicagent. In some embodiments, the treatment is a preventative treatment.

In the practice of the foregoing methods, the subject to which thedietary supplement or therapeutic composition is administered may be asubject in need of renal health support, for example, a subject havingor at risk of a renal condition. A renal condition, as used herein, mayencompass any pathological process, pathological state, disease, ordysfunction of the kidney. In various embodiments, for example, thesubject is a human, a cat, or a dog.

In various embodiments, the renal condition is a cystic condition. Inone embodiment, the cystic condition is PKD. In various embodiments,supporting renal health and/or treatment of a cystic conditionsencompasses preventing or ameliorating any pathological effects of renalcysts, reducing or preventing the formation of renal cysts, reducing thenumber and/or size of renal cysts, slowing the growth rate of renalcysts, or eliminating renal cysts. In one embodiment, the cysticcondition is ADPKD. In one embodiment, the polycystic kidney disease isautosomal recessive polycystic kidney disease (ARPKD). In variousembodiments, the subject to which the dietary supplement or therapeuticcomposition is administered is a subject having PKD or at risk of PKD.In some embodiments, the subject at risk of PKD is a subject carrying amutation in any of PKD1, PKD2, PKD3, or other genes wherein mutationsincrease the risk of PKD. In some embodiments, the subject at risk ofPKD is a subject with health or demographic factors associated with PKDrisk. In some embodiments, the subject is a human. In some embodiments,the subject is a dog or cat.

In other embodiments, the renal cystic condition is a condition selectedfrom the group consisting of renal cysts as found in tuberous sclerosiscomplex, medullary cystic kidney disease, Acquired Cystic KidneyDisease, including acquired simple renal cysts, cystic renal cellcarcinoma, and cystic nephroma.

In one embodiment, the renal condition is acute kidney injury (AKI). AKIencompasses kidney dysfunction or failure, and may be caused by manyfactors or causative agents. AKI manifests as damage to kidneys andimpaired kidney functions, including progression along the RIFLE stagingcriteria known in the art, encompassing risk, injury, failure, loss, andend-stage renal disease. In various embodiments, the subject to whichthe dietary supplement or therapeutic composition is administered is asubject having or at risk of AKI. In various embodiments, a subject atrisk of AKI is a subject having systemic disease, trauma, sepsis, renalartery stenosis, renal vein thrombosis, urinary tract obstruction,glomerulonephritis, acute tubular necrosis, acute interstitialnephritis, or liver cirrhosis. In various embodiments, a subject at riskof AKI is a subject having reduced blood flow to kidneys, such as bycardiac dysfunction or failure, surgery, trauma or ischemia. In variousembodiments, a subject at risk of having AKI is a subject likely to beexposed to or being exposed to harmful levels of nephrotoxins such asmercury or platinum compounds, radiological contrast agents, andantibiotics. In one embodiment, the subject at risk of AKI is a subjectlikely to have or scheduled to have surgery, in one embodiment, cardiacsurgery. In various embodiments, dietary supplement and/or therapeuticcomposition is administered as a pre-treatment prior to surgery and/orpost-treatment after surgery in order reduce the risk or severity of AKIand to reduce the risk of developing complications therefrom, includingchronic kidney disease.

In various embodiments, the renal condition is chronic kidney disease(CKD), encompassing any gradual loss or long-term impairment of kidneyfunction. In various embodiments, the subject to which the dietarysupplement or therapeutic composition is administered is a subjecthaving or at risk of CKD. A subject at risk of CKD may be a subjecthaving any known risk factor of CKD including, Type I or Type IIdiabetes mellitus, prior occurrence of acute kidney injury,glomerulornephritis, high blood pressure, or family history of CKD.

In various embodiments, the renal condition is a condition selected fromthe group consisting of: diabetic nephropathy, nephronophthisis,Meckel-Gruber syndrome, Bardet-Biedl syndrome, Joubert syndrome,medullary sponge kidney, multicystic dysplastic kidney, Dent's disease,Glomerulocystic kidney disease, Von Hippel-Lindau Syndrome, and mixedepithelial and stromal tumor of the kidney.

Methods of Supporting Health and Treating Reperfusion Injury. In anotheraspect, the methods of the invention are directed to various conditionsdescribed as ischemic reperfusion injuries (IRI), sometimes referred toas reoxygenation injury. This class of pathologies is associated withthe loss of blood flow in the affected organ, structure, or area. Thehypoxia and loss of nutrients in the affected area promotes conditionswherein the subsequent restoration of circulation results ininflammation and damage caused by severe oxidative stress. The loss ofcirculation may be the result of trauma, injury, ischemic events,cardiac failure, or other causes. Surgery is also a causative agent ofIRI, especially cardiac surgery, wherein reduced blood flow during theprocedure facilitates IRI upon restoration of normal blood flow. IRI mayaffect any number of tissues, including the arteries and veins, brain,heart, liver, lungs, kidneys, skeletal muscle, and digestive tract.

In one implementation, the scope of the invention encompasses a dietarysupplement comprising a ketonic composition for use in a method ofsupporting health with regards to ischemic reperfusion injury. In arelated embodiment, the scope of the invention encompasses the use of aketonic composition in a method of making a medicament for the supportof health with regards to ischemic reperfusion injury. In a relatedimplementation, the scope of the invention encompasses a method ofsupporting health with regards to ischemic reperfusion injury in asubject by the administration to the subject (for example, includingself-administration, i.e. ingestion) of a biologically effective amountof a dietary supplement comprising a ketonic composition. In variousembodiments, the dietary supplement may comprises a ketonic agent incombination with a crystal precipitation inhibitor. In one embodiment,supporting health with respect to ischemic reperfusion injury meansmaintaining or enhancing one or more measures of organ function, forexample, function of the brain, heart, liver, lungs, kidneys, skeletalmuscle, or digestive tract. In one embodiment, supporting health withrespect to ischemic reperfusion injury in a subject is achieved byameliorating the subject's risk, progression, and/or severity of one ormore ischemic reperfusion injury conditions.

In another aspect, the scope of the invention encompasses the use oftherapeutic compositions comprising ketonic compositions for thetreatment of ischemic reperfusion injury conditions. In one embodiment,the scope of the invention encompasses a ketonic composition for use ina method of treating an ischemic reperfusion injury condition. In arelated embodiment, the scope of the invention encompasses the use of aketonic composition in a method of making a medicament for the treatmentof ischemic reperfusion injury. In one embodiment, the scope of theinvention encompasses a method of treating a ischemic reperfusion injurycondition in a subject in need of treatment therefor by administering tothe subject a therapeutically effective amount of a ketonic composition.The ketonic composition may be delivered or provided in a therapeuticcomposition, for example, a formulation comprising other componentsand/or active ingredients. In some embodiments, the therapeuticcomposition comprises a ketonic composition in combination with acrystal precipitation inhibitor. In some embodiments, the treatment is apreventative treatment.

In the foregoing methods of the invention, the subject to which thedietary supplement or therapeutic composition comprising a ketoniccomposition is administered is a subject in need of treatment for one ormore ischemic reperfusion injuries, in some embodiments being a subjectthat has experienced, or is at risk of the one or more ischemicreperfusion injuries. In one embodiment, the subject at risk of ischemicreperfusion injury is a subject likely or scheduled to undergo surgery.In one embodiment, the dietary supplement or therapeutic composition isadministered as a pre-treatment prior to surgery.

Methods of Supporting Neurological Health and Treating NeurodegenerativeConditions. In various implementation, the methods of the invention aredirected to maintaining or improving some aspect of neurological health,and to addressing neurological conditions, including conditions of thebrain, central nervous system, and peripheral nervous system.

In one implementation, the scope of the invention encompasses a dietarysupplement comprising a ketonic composition for use in a method ofsupporting neurological health. In a related implementation, the scopeof the invention encompasses a method of supporting neurological healthin a subject by the administration to the subject (for example,including self-administration, i.e. ingestion) of a therapeuticallyeffective amount of a dietary supplement comprising a ketoniccomposition. In one embodiment, supporting neurological health meansmaintaining or enhancing one or more measures of neurological function,for example, cognitive function or motor function. In one embodiment,supporting neurological health in a subject is achieved by amelioratingthe subject's risk, progression, and/or severity of one or moreneurological conditions.

In another implementation, the scope of the invention encompassestherapeutic compositions comprising a ketonic composition for use in amethod of treating a neurological condition. In a related embodiment,the scope of the invention encompasses the use of a ketonic compositionin a method of making a medicament for treatment of a neurologicalcondition. In a related embodiment, the scope of the inventionencompasses a method of treating a neurological condition in a subjectin need of treatment therefor, comprising administering to the subject atherapeutically effective amount of a therapeutic composition comprisinga ketonic composition. In some embodiments, the therapeutic compositionfurther comprises a crystal precipitation inhibitor and/or a lipotoxicagent. In some embodiments, the treatment is a preventative treatment.

In the foregoing methods, the subject to which the dietary supplement ortherapeutic composition is administered may be a subject in need oftreatment for one or more neurological conditions. A neurologicalcondition, as used herein, may encompass any pathological process,pathological state, disease, or dysfunction of the central or peripheralnervous system, especially conditions that affect the brain, forexample, a neurodegenerative disease. In various embodiments, thesubject at risk of a neurological condition is has, or is at risk of theone or more neurological conditions. In one embodiment, the neurologicalcondition is a neurodegenerative condition. In various embodiments, theneurological condition is selected from the group consisting ofAlzheimer's disease, Parkinson's disease, multiple sclerosis,amyotrophic lateral sclerosis, Huntington's disease, epilepsy, chronictraumatic encephalopathy, age-related neurodegeneration, frontotemporaldementia, and retinitis pigmentosa.

Methods of Supporting Cilial Health and Treating Ciliopathies. In someembodiments, the methods of the invention are directed to health aspectsand conditions associated with the cilia. Cilia are a component ofalmost all vertebrate cells. Ciliopathies encompass a large group ofdisorders associated with cilial dysfunction, typically driven bygenetic mutations encoding defective proteins which result in eitherabnormal formation of cilia or which disrupt their normal function.Ciliopathies include a wide array of conditions across various organsand systems of the body.

In one implementation, the scope of the invention encompasses a dietarysupplement comprising a ketonic composition for use in a method ofsupporting cilial health. In a related embodiment, the scope of theinvention encompasses the use of a ketonic composition in a method ofmaking a medicament for the support of cilial health. In a relatedimplementation, the scope of the invention encompasses a method ofsupporting cilial health in a subject by the administration to thesubject (for example, including self-administration, i.e. ingestion) ofa biologically effective amount of a dietary supplement comprising aketonic composition. In one embodiment, supporting cilial health meansmaintaining or enhancing one or more measures of cilial function. In oneembodiment, supporting cilial health in a subject is achieved byameliorating the subject's risk, progression, and/or severity of one ormore ciliopathies.

In another implementation, the scope of the invention encompasses atherapeutic compositions comprising a ketonic composition for use in amethod of treating a ciliopathy. In a related embodiment, the scope ofthe invention encompasses the use of a ketonic composition in a methodof making a medicament for the treatment of a ciliopathy. In a relatedembodiment, the scope of the invention encompasses a method of treatinga ciliopathy in a subject in need of treatment therefor, comprisingadministering to the subject a therapeutically effective amount of atherapeutic composition comprising a ketonic composition. In someembodiments, the therapeutic composition further comprises a crystalprecipitation inhibitor and/or a lipotoxic agent. In some embodiments,the treatment is a preventative treatment to a subject at risk of aciliopathy.

In the foregoing methods, the subject to which the dietary supplement ortherapeutic composition is administered may be a subject in need oftreatment for one or more ciliopathies, in some embodiments being asubject that has, or is at risk of the one or more ciliopathies. Invarious embodiments, the ciliopathy is selected from the groupconsisting of polycystic kidney disease, polycystic liver disease,retinitis pigmentosa, congenital fibrocystic diseases of the liver,diabetes, obesity, skeletal dysplasia's, Ahlstrom syndrome, Bardet-Biedlsyndrome, Joubert syndrome, nephronophthisis, orofaciodigital syndrome1, Senior-Loken syndrome, primary ciliary dyskinesia, Jeune asphyxiatingthoracic dysplasia, Marden-Walker syndrome, situs inversus or isomerism,retinal degeneration, agenesis of the corpus callosum, anencephaly,cerebellar vermis hypoplasia, Dandy-Walker malformation, diabetes,Ellis-van Creveld syndrome, exencephaly, eye movement abnormalities,hypoplasia of the corpus callosum, hypotonia, sterility, Jeuneasphyxiating thoracic dystrophy, Juvenile myoclonic epilepsy,Marden-Walker syndrome, Meckel-Gruber syndrome, sensorineural deafness,and spina bifida.

Other Uses of Dietary Supplements. In various embodiments, the dietarysupplements and therapeutic compositions comprising ketonic agents maybe utilized in methods of preventing or treating conditions such ascancer, a cystic condition of the liver, a pulmonary disease, autoimmunedisease, a condition of the digestive tract, epilepsy, or infection.

In one embodiment, the dietary supplements and therapeutic compositionsof the invention are used in the treatment of viral infection. It hasbeen shown in the art that ketosis may increase the body's ability tofight viral infections, for example, as described in Goldberg et al.,Ketogenic diet activates protective γδ T cell responses againstinfluenza virus infection. Science Immunology, 2019; 4 (41): eaav2026.In one embodiment, the scope of the invention encompasses a dietarysupplement comprising a ketonic composition for use in a method ofpreventing or treating viral infection. In one embodiment, the scope ofthe invention encompasses a method of treating a viral infection, forexample, preventing viral infection, in a subject by the administrationto the subject of a therapeutically effective amount of a therapeuticcomposition comprising a ketonic composition. The viral infection may aninfection caused by any pathogenic virus, for example influenza viruses,coronaviruses such as COVID19, HIV, noroviruses, respiratory syncytialviruses, herpes viruses, and papillomaviruses.

EXAMPLES Example 1 Dietary Ketosis and Exogenous BHB Ameliorate RenalCyst Growth in Polycystic Kidney Disease

A key signaling molecule activated in ADPKD is the kinase mammaliantarget of rapamycin (mTOR) that regulates many cellular behaviorsincluding proliferation, cell growth, and energy metabolism and isresponsive to growth factors, cellular energy status, and nutrientavailability. mTOR inhibitors, such as rapamycin, were highly effectivein PKD rodent models but failed in subsequent clinical trials, mostlikely due to dose-limiting, significant extra-renal side effects andtoxicities.

As an alternative to pharmacological intervention, modulation of mTORactivity in the kidney via dietary intervention was explored, since mTORis under control of nutrient availability. As reported in Kipp, et al.(2016). A mild reduction of food intake slows disease progression in anorthologous mouse model of polycystic kidney disease. Am. J. Physiol.Renal Physiol. 310, F726-F731, a very mild reduction in food intake, byonly 23%, profoundly inhibited renal cyst growth and mTOR activity whilenot affecting body weight gain. Another study, using two differentorthologous mouse models, had similar conclusions and showed that foodrestriction by 40%, and even by only 10%, significantly inhibited renalcyst growth, as reported in Warner, et al., (2016). Food restrictionameliorates the development of polycystic kidney disease. J. Am. Soc.Nephrol. 27, 1437-1447. These results raise questions about themechanism underlying the beneficial effect of dietary restriction,including whether the inhibition of renal cyst growth was due to overallcaloric restriction or due to the restriction of a particular macro- ormicro-nutrient, or by a different mechanism entirely.

As set forth herein, dietary restriction strongly inhibits renal cystgrowth due to metabolic changes caused by intermittent fasting.Time-restricted feeding, in comparison to isocaloric ad libitum feeding,causes an intermittent decrease of blood glucose and an increase inketogenesis and leads to strong inhibition of renal cyst growth,proliferation, and fibrosis. These effects are not merely due tocircadian feeding rhythm because ad libitum administration of aketogenic diet (KD) similarly inhibits renal cyst growth. Herein isshown that acute fasting in mouse, rat, and feline models of PKD inducessignificant apoptosis in cyst-lining epithelial cells and a strikingreversal of renal cystic burden. Finally, it is shown herein,surprisingly, that supplementation of diet with just the natural ketoneBHB replicates the beneficial effects of dietary restriction. Theseresults indicate that dietary restriction has profound inhibitoryeffects on PKD progression, and this depends on induction of ketosis, asrenal cyst cells in PKD appear to be metabolically inflexible and thusunable to adapt to alternative fuel sources. These results demonstratethat disease progression in ADPKD can be controlled by dietaryinterventions such as time-restriction or KDs and also, unexpectedly, byBHB supplementation or treatments that replicate BHB action.

Glucose Levels Predict the Rate of Kidney Volume Change in Individualswith ADPKD. Individuals with ADPKD and type 2 diabetes havesignificantly larger total kidney volume (TKV) than those with ADPKDalone, and overweight or obesity associates with faster progression inearly-stage ADPKD. To investigate whether serum glucose levels even innormoglycemic, serum glucose was explored in a cohort of human ADPKDsubjects with preserved renal function. Baseline serum glucose in thiscohort uniquely predicted a greater TKV and htTKV increase over time.Similar analyses found no effect of the baseline serum glucose onaverage yearly change in GFR. Together, these results suggest thatglucose availability may directly affect renal cystic diseaseprogression in ADPKD and that limiting glucose availability mayameliorate cystic progression in PKD.

Han:SPRD rats, a non-orthologous model of PKD with a mutation in Anks6were placed on a time-restricted feeding (TRF) regime wherein TRFanimals had access to food for an 8-h period within their 12-h darkcycle. Animals were treated for 5 weeks from post-natal weeks 3-8. TRFanimals consumed a comparable number of calories and gained weightsimilar to the AL controls, indicating that TRF does not lead to food orcaloric restriction, and there were no negative effects measured on anyother organ. After 5 weeks of this dietary regimen, animals in the TRFcohort exhibited strikingly reduced renal cystic disease progressioncompared to animals in the AL cohort. Kidneys of TRF-treated animalswere significantly less cystic (FIG. 2A) with a marked reduction in the2-kidney/body weight ratio and improved kidney function as indicated bynormalization of serum creatinine. In whole kidney sections, total cystnumber (FIG. 2B) and cyst size (FIG. 2C), were decreased in TRF animalscompared to controls, demonstrating that TRF inhibited both cystogenesisand cyst expansion. Additionally, TRF-treated animals had increasedlevels of BHB and reduced blood glucose indicative of ketosis.

TRF Reduces mTORC1 and STAT3 Signaling, Interstitial Fibrosis, andProliferation in Cystic Kidneys. Changes in known PKD associatedpathways and pathologies were assessed. A significant reduction inphosphorylated S6^(S235/236) (FIG. 3) a downstream target in the mTORpathway, was observed in cyst-lining epithelial cells of TRF-treatedmale, but not female, Cy/+ animals indicating inhibition of mTORC1activity. PKD is known to lead to interstitial fibrosis, a hallmark ofprogressive renal disease. Collagen deposition was markedly reduced inTRF compared to AL animals. Myofibroblasts were strongly reduced andfrequently absent in kidneys of TRF animals. Staining with the cellcycle marker Ki-67 revealed a significant reduction in cyst-lining cellsin TRF compared to AL animals, indicating inhibition of proliferation.Active, phosphorylated STAT3, which has previously been shown to beactivated in PKD and drives cystic progression, was reduced or absent incysts in TRF-treated animals.

A KD Ameliorates Disease Progression in Juvenile Han:SPRD Rats. Han:SPRDrats were given ad libitum access to a high-fat, very-low-carbohydrateketo diet (KD-caloric ratio of 91% fat, 2% carbohydrates, and 5%protein) and compared to animals with ad libitum access to NC (caloricratio of 62% carbohydrates, 25% protein, and 13% fat). Animals weretreated again for 5 weeks from post-natal weeks 3-8. Renal cysticdisease progression was strongly inhibited in animals on the KD regimencompared to those on NC along with a remarkable reduction in the2-kidney to body weight and cystic area (FIG. 4A). KD-treated ratsexhibited decreased blood glucose and increased BHB levels compared toNC controls, and the level of ketosis was more profound than thatachieved with the TRF regimen. Animals on the KD appeared phenotypicallynormal and did not exhibit any signs of distress during the study.However, KD-treated animals exhibited reduced body weight gain, and onlygained approximately 2%-10% of their body weight each week during thestudy. This lack of growth was not dependent on caloric intake, as theKD group consumed comparable calories to NC controls relative to theirbody weight, likely resulting from protein restriction. KD had adisproportionate inhibitory effect on the growth of poly-cystic kidneysas compared to normal kidneys. Importantly, KD feeding led to improvedkidney function (FIG. 4B) as well as inhibited both cystogenesis (FIG.4C) and cyst expansion (FIG. 4D).

The effects of KD treatment on PKD progression were generally similar tothose caused by the TRF treatment. mTOR (FIG. 5) and STAT3 activity incystic epithelia were both blunted in KD-fed rats. SMA1-positivemyofibroblasts were less in KD-fed animals compared to NC controls andwas accompanied by a decrease in collagen deposition and the percent ofKi-67-positive demonstrating inhibition of cell cycle entry andproliferation (FIG. 6A). It was also found that KD feeding increased thelevels of phospho-AMPK in the kidneys of cystic animals and increasedCPT1a levels in male cystic, but not female cystic, rats (FIGS. 6B and6C).

A KD Ameliorates Disease Progression in Adult Han:SPRD Rats. Han:SPRDrats at an older age (post-natal week 8-12) were fed KD versus NC. Atthis age, there is no longer cystic kidney enlargement, and no furtherchange in kidney size was observed. During the treatment period, weightgain was mildly affected in rats on the KD, and there was no differencebetween wild-type and cystic animals. In contrast, the total mass ofpolycystic kidneys remained nearly the same in animals on NC but loweredby 35% in male rats on KD, accompanied by diminished 2-kidney to bodyweight ratio and percent cystic area (FIG. 6A). Similar to the juvenileexperiments, the KD again affected the organ size of polycystic kidneysdisproportionally because wild-type kidneys or other organs, such as theheart, were largely unaffected. Unlike the juvenile experiments, serumcreatinine was unaffected by KD treatment in adult rats as was the totalnumber of cysts per animal (FIG. 6B). There was, however, a markedreduction in cyst size (FIG. 6C), indicating that KD feeding preventscyst growth in animals at that age. KD feeding led to a reduction infibrosis in male animals, as indicated by reduced collagen, but therewas no detectable difference in myo-fibroblasts, mTOR activity, or thecell cycle marker Ki-67.

Acute Fasting Leads to Cell Death and Renal Cyst Regression in MultiplePKD Animal Models. The strong inhibitory effects on PKD progression wasobserved with ketosis induced by TRF and KD regimens during the courseof several weeks. To investigate whether ketosis has any fast-actingeffects on renal cysts, Han:SPRD rats were subjected to acute ketosisvia fasting. 8-week-old animals were fasted for 48 h with free access towater. As expected, acute fasting led to decreased blood glucose andincreased blood BHB values (FIGS. 7A and 7B), indicating the inductionof ketosis. The fasting-induced modest decrease in body weight (12%) isexpected due to depletion of fat and glycogen reserves, primarily inadipose tissue, liver, and skeletal muscle, respectively, and wasconsistent between wild-type and cystic animals. Strikingly, acutefasting strongly affected cystic kidneys, leading to a 20% reduction incystic area (FIG. 7C) and a concurrent reduction in kidney mass and the2-kidney/heart weight ratio. Fasting had minimal-to-no effect on theweight of hearts and normal kidneys as expected, indicating that thefasting effects are specific to PKD kidneys. The loss of mass was notdue to any loss of glycogen, and the reduction of cystic volumeaccounted for the observed loss in kidney mass, demonstrating that acutefasting leads to a loss of cyst fluid.

TUNEL staining revealed that acute fasting led to greater cell death ofcystic cells but did not cause greater cell death in normal, non-cystickidneys. In particular, there was a conspicuous number ofTUNEL-positive, detached cells in cyst lumens, indicating that thesedead cells originated from sloughed-off cyst-lining cells. Renal cystsin fasted animals frequently exhibited fragmented cells and denudedepithelium, which was not observed in renal cysts of control-fed cysticanimals, demonstrating that loss of cyst fluid caused by acute fastingis due to cell death and disruption of the epithelial barrier of cysts,leading to draining of cyst fluid, presumably via the interstitium andlymphatics, leading to overall shrinking of polycystic kidneys.

Next was explored the possibility that increased supply of fatty acidsduring ketosis may affect cystic cells. As expected, intracellular oildroplets are rarely observed in normal or cystic kidneys in control-fedanimals while acute fasting leads to accumulation of oil droplets intubule cells in normal kidneys consistent with previous reports.Cyst-lining cells in polycystic kidneys exhibited a much exaggerateddegree of cytoplasmic oil droplet accumulation. Frequently, most of thecytoplasm of the affected cells appeared to be occupied by oil dropletssuggesting a state of severe steatosis. These findings demonstrate thatcyst-lining cells have the ability to take up circulating fatty acidsduring acute fasting but are unable to sufficiently metabolize them,leading to lipotoxicity and greater cell death.

To test acute ketosis in an orthologous mouse model of PKD, theorthologous Pkdl:Nestin-Cre model was used. Fasting for 24 h, with freeaccess to water, led to a pronounced decrease in blood glucose andincrease in blood BHB but no significant change in kidney mass or2-kidney to body weight. TUNEL staining revealed that acute fastinginduced apoptotic cell death in cyst-lining cells and greaterTUNEL-positive luminal cells, in polycystic kidneys but not in normalkidneys. It is speculated that the short duration of fasting in mice isinsufficient to lead to significant cyst draining and a measurableeffect on cystic burden, as compared to rats.

Oral BHB Treatment Prevents Polycystic Disease Progression in JuvenileRats. To investigate if BHB might affect PKD progression, Han:SPRD ratswere treated with BHB from post-natal weeks 3-8 in the context of adlibitum feeding with normal, high-carbohydrate chow (NC). BHB wasadministered as a sodium and potassium salt in the drinking water adlibitum at a dose equivalent to that recommended as a dietary supplementin BHB supplement drinks. Control cohorts received either normal wateror water supplemented with the molar equivalents of salt provided in theBHB supplement (a mixture of NaCl/KCl). All treated animals consumedcomparable amounts of water and calories, and BHB treatment had noeffect on body weight or blood glucose levels. Surprisingly, after 5weeks of treatment with BHB, the kidneys of PKD animals were nearlyindistinguishable from kidneys of wild-type animals, both at the grossand histological level. BHB-treated PKD rats showed striking andunexpected reductions in the 2-kidney to body weight ratio and thecystic area (FIG. 8A) compared to water- and salt-treated controls.Neither wild-type kidneys nor other organs were affected by BHBtreatment, indicating that the effect is specific to polycystic kidneys.Surprisingly, BHB treatment led to a strong reduction in fibrosis,almost complete elimination of myofibroblasts, improved kidney function(assessed by creatinine), and inhibition of proliferation.

These unexpected results demonstrate that BHB acts in a dominant fashionas a suppressor of PKD progression, even in animals that are fed ahigh-carbohydrate diet and have unaltered blood glucose levels.Altogether, our results demonstrate that inducing a state of ketosis,either by dietary intervention or surprisingly even by mimicking itseffects with BHB, is an effective treatment for ADPKD in humans. Acommon factor in all of the interventions described herein was theelevation of BHB. Remarkably, treatment with BHB alone, even in thecontext of a normal high-carbohydrate rodent diet proved to be highlyeffective. Unexpectedly, this demonstrates that BHB itself is a majorfactor in suppressing PKD progression.

The results also demonstrate that ketosis strongly inhibits not onlyrenal cyst growth but also fibrosis. During the progression of PKD, cystproliferation and fibrosis typically go hand in hand, and it is possiblethat both processes may reinforce each other. Myofibroblasts have beenshown to be abundant in PKD kidneys and are thought to be a majorcontributor to fibrosis. Herein was demonstrated that ketosis greatlyand unexpectedly reduces the abundance of pericystic myofibroblasts,especially the TRF regimen and BHB treatment, to almost completeabsence. Consequently, overall fibrosis, as measured by collagendeposition, is strongly reduced.

Most individuals with ADPKD in industrialized societies consume ahigh-carbohydrate diet throughout their waking hours and only rarely, ifever, experience periods of ketosis. A recent nutritional feasibilitystudy recommends high-carbohydrate diets for individuals with ADPKD, asreported in Taylor, et al. (2017). Diet and polycystic kidney disease: apilot intervention study. Clin. Nutr. 36, 458-466, as do many availablecookbooks, websites, online forums, etc., where individuals with ADPKDmay seek dietary advice. The results provided herein suggest that suchdietary habits may worsen PKD progression. In contrast, moderation incaloric intake has been recommended for the management of ADPKD, asreported in Chebib, and Torres (2018). Recent advances in the managementof autosomal dominant polycystic kidney disease. Clin. J. Am. Soc.Nephrol. 13, 1765-1776. The results presented herein, however,demonstrate that reducing caloric intake per se would not necessarilylead to inhibition of cystic progression but that the induction of thestate of ketosis is instead important, irrespective of caloric intake.

Example 2 Co-Administration of BHB and Citrate Ameliorate Renal CystGrowth in Polycystic Kidney Disease

To investigate BHB, citrate, and combined BHB and citrate effects on PKDprogression, Han:SPRD rats were treated with BHB or a combination of BHBand citrate from post-natal weeks 3-8 in the context of ad libitumfeeding with normal, high-carbohydrate chow (NC). In the BHB treatments,BHB was administered as a sodium and potassium salt in the drinkingwater ad libitum at a dose equivalent to that recommended as a dietarysupplement in BHB supplement drinks. In the citrate treatments, citratewas administered as a combination of tripotassium citrate and citricacid at a (0.82/1) ratio at a dose equivalent to that recommendedutilized in citrate dietary supplements. Control cohorts received eithernormal water or water supplemented with the molar equivalents of saltprovided in the BHB or BHB-citrate supplements (a mixture of NaCl/KCl).Control cohorts received either normal water or water supplemented withthe molar equivalents of salt provided in the BHB or BHB-citratesupplements (a mixture of NaCl/KCl). All treated animals consumedcomparable amounts of water and calories, and BHB treatment had noeffect on body weight or blood glucose levels. After 5 weeks oftreatment with BHB or BHB citrate, the kidneys of PKD animals werenearly indistinguishable from kidneys of wild-type animals both at thegross and histological level. BHB or BHB-citrate treated PKD rats showedstriking reductions in the 2-kidney to body weight ratio (FIG. 8A) andthe cystic area (FIG. 8B) compared to water- and salt-treated controls.Neither wild-type kidneys nor other organs were affected by BHBtreatment, indicating that the effect is specific to polycystic kidneys.

The results demonstrate dose-dependent benefits of BHB, and similardose-dependent therapeutic effects from citrate, particularly at the 120mM dosage. These results also demonstrate that the combination of BHBwith citrate provides a synergistic response in PKD rats, even achievedat lower dosages of both BHB and citrate.

Example 3 GPR109A Activation in PKD

The results described above led to the unexpected conclusion that BHBhas a strikingly strong dominant inhibitory effect on PKD progression.The most immediate and likely assumption would be that BHB may act byvirtue of its property as a metabolite that is taken up by cells andused for energy generation. However, further experimentation led tounexpected results suggesting that this model is incorrect. Instead, theresults surprisingly suggest that the beneficial action of BHB as aninhibitor of PKD progression is due to the activation of a specificreceptor termed GPR109A. GPR109A is a member of the class of G-proteincoupled receptors. It has recently been identified as the niacinreceptor and it mediates the lipid-lowering effects of niacin due to itsactivation on adipocytes. It has been found that GPR109A is not onlyactivated by niacin but also by high concentrations of BHB such as mayoccur during fasting-induced ketosis. The inventors of the presentdisclosure speculated that GPR109A may be expressed on cells in thekidney and that treatment with BHB would lead to its activation andaffect PKD progression. To test this, GPR109A knockout mice were crossedwith a PKD model. In these mice, the loss of GPR109A surprisingly indeedled to accelerated PKD progression, indicating that GPR109A is normallya suppressor if cyst growth. This is a novel finding that would havebeen unanticipated without the discoveries presented herein regardingthe beneficial effect of BHB in ameliorating PKD progression.Additionally, treatment with niacin, a GPR109A agonist, ameliorates PKDprogression in PKD mice expressing GPR109A but not in GPR109A knockoutmice. For example, as presented in FIG. 9, PKD progression (as assessedby 2-kidney weight to total body weight ratio, 2KW/TBW) was amelioratedby niacin treated PKD mice (10 mg/kg, daily ip, from postnatal day 8-21)that express GPR109A (Genotype 1), while niacin treatment had nobeneficial effect on PKD progression in the GPR109A knockout mice(Genotype 2).

All patents, patent applications, and publications cited in thisspecification are herein incorporated by reference to the same extent asif each independent patent application, or publication was specificallyand individually indicated to be incorporated by reference. Thedisclosed embodiments are presented for purposes of illustration and notlimitation. While the invention has been described with reference to thedescribed embodiments thereof, it will be appreciated by those of skillin the art that modifications can be made to the structure and elementsof the invention without departing from the spirit and scope of theinvention as a whole.

What is claimed is:
 1. A composition, comprising a mixture of: citrate;and BHB and/or BHB analog; wherein the weight percentage of citrate isat least about 20%, calculated as the proportional mass of citrate tothe combined mass of BHB and/or BHB analog and citrate.
 2. Thecomposition of claim 1, wherein the BHB and/or BHB analog comprises BHB.3. The composition of claim 1, wherein the BHB and/or BHB analogcomprises a BHB analog.
 4. The composition of claim 1, wherein theweight percentage of citrate is in the range of about 35% to about 45%.5. The composition of claim 1, wherein the weight percentage of citrateis about 40%.
 6. The composition of claim 1, wherein the composition isformulated as a powder to be dissolved in liquid.
 7. The composition ofclaim 1, wherein the composition is provided in a dosage formcomprising: at least about 1.0 g citrate; and about 2.0-3.0 g BHB. 8.The composition of claim 1, wherein the composition is formulated asinfused water, a sports drink, an energy drink, tea, fruit juice, or ameal replacement beverage.
 9. The composition of claim 1, wherein thecomposition is formulated as a food item.
 10. The composition of claim1, wherein the food item is formulated for non-human animals andcomprises chow, feed, a treat or snack, or supplemented pet food. 11.The composition of claim 1, wherein the composition is formulated as adietary supplement.